Copper-based grape pest management has impacted wine aroma

Despite the high energetic cost of the reduction of sulfate to H2S, required for the synthesis of sulfur-containing amino acids, some wine Saccharomyces cerevisiae strains have been reported to produce excessive amounts of H2S during alcoholic fermentation, which is detrimental to wine quality. Surprisingly, in the presence of sulfite, used as a preservative, wine strains produce more H2S than wild (oak) or wine velum (flor) isolates during fermentation. Since copper resistance caused by the amplification of the sulfur rich protein Cup1p is a specific adaptation trait of wine strains, we analyzed the link between copper resistance mechanism, sulfur metabolism and H2S production. We show that a higher content of copper in the must increases the production of H2S, and that SO2 increases the resistance to copper. Using a set of 51 strains we observed a positive and then negative relation between the number of copies of CUP1 and H2S production during fermentation. This complex pattern could be mimicked using a multicopy plasmid carrying CUP1, confirming the relation between copper resistance and H2S production. The massive use of copper for vine sanitary management has led to the selection of resistant strains at the cost of a metabolic tradeoff: the overproduction of H2S, resulting in a decrease in wine quality.

The most ancient traces of wine making have been discovered in Georgia 1 and have been dated as 6000 BC.Since that ancient time, cultivation of grapevine and winemaking knowledge spread progressively all over the world 2 .All along this period, winemaking practices have evolved, especially with the discovery of the use of sulfite to limit the growth of undesired microorganisms to protect wine from oxygen and to preserve aroma profile.Similarly, the cultivation of Vitis vinifera has faced changes, especially with the development of grafting and the spray of chemical compounds required to face the import in Europe of three major pests for vine: phylloxera, powdery mildew and downy mildew.Among chemicals sprayed on vines, copper has been intensively used in vineyards to control the development of Plasmopara viticola.This intensive use of copper in vineyards has translated into high copper in grape musts 3 .
Wine fermentation is mainly achieved by the yeast species Saccharomyces cerevisiae which is found also in many fermented products: sake, bread, cheese and more [4][5][6][7] , as well as in natural biotopes such as forests 8,9 .S. cerevisiae strains display specific physiological properties associated to the different ecological niches they live in, as result of several domestication events 4,[10][11][12] .
One of the most remarkable and contrasting adaptation events can be seen in fermenting wine strains and in wine velum isolates (flor yeasts).S. cerevisiae velum strains have developed a specialized aerobic lifestyle, highly different from the one of fermenting wine strains 13 .Since they colonize the wine when fermentation is concluded, velum strains develop the ability to grow in media depleted for nitrogen, vitamins, glucose and fructose.
Wine fermentation poses a challenging environment for S. cerevisiae.Different genomic features have been identified as traces of adaptation to the wine environment, in line with its domestication 14 .The first and best described adaptation of S. cerevisiae to the wine environment is the resistance to sulfite, obtained from several translocation events resulting in a high expression of the sulfite export pump Ssu1 [15][16][17][18][19] .Another example of adaptation to the grape and must environments can be seen in the selection of strains carrying multiple copies of the CUP1 gene.This gene amplification leads to an enhanced protein abundance/synthesis, providing resistance to high concentrations of copper in the grape must, resulting from the massive use of copper as fungicide 20 .Cup1p is among the ten sulfur richest yeast proteins 21 and some S. cerevisiae strains can harbor up to 79 copies 18,20,22 .Therefore, the high synthesis of Cup1p caused by its amplification requires a high availability of sulfur containing amino acids methionine and cysteine that are scarce in grape musts.These amino acids can be synthesized by yeast through the sulfur assimilation pathway (SAP), which reduces inorganic sulfate into hydrogen sulfide (H 2 S) with the consumption of 7 mol of NADPH and 4 of ATP per mole of S-amino acid 23 .Consequently, the biosynthesis of the sulfur amino acids has a significant impact on the yeast redox and energy balances.A high diversity in the production of H 2 S during alcoholic fermentation has been described for wine strains 24 , and because its content is detrimental to wine aroma, different studies have deciphered its genetic bases and found allelic variations in MET10, SKP2, MET2, TUM1 [25][26][27][28] , genes involved in the sulfur assimilation pathway or its regulation.Some of these findings have been patented and have led to the improvement of industrial winemaking starters.Surprisingly, no investigation has been carried out to understand the biological meaning of such overproduction, nor to evaluate a potential relation with different ecological niches.Interestingly, for wine S. cerevisiae, SO 2 and copper tolerance have been found negatively associated 29 .Transcriptional and proteomic analysis in sulfur-limited medium, demonstrated that SSU1 over-expression induced sulfur limitation during exposure to copper and provoked an increased sensitivity to copper 30 .
Because the production of H 2 S is so costly to the cell 23 , we wondered why some wine strains were overproducing it.Comparing three groups of strains: isolated from velum and wine, two contrasted anthropogenic environments, and oak, as a natural environment, we show that the total content of H 2 S produced during alcoholic fermentation depends on the ecological niche, and that exposure of yeast cells to copper enhances H 2 S production.We evaluated how the amplification of CUP1 may explain such variation, using a set of strains with variable number of copies of CUP1 or strains carrying a plasmid overproducing CUP1.Last, we measured the impact of sulfites availability in the media on copper resistance.

Strain variability in H 2 S production during alcoholic fermentation
To assess the variability of the production of H 2 S during alcoholic fermentation of Saccharomyces cerevisiae, we evaluated 33 strains isolated from three ecological niches: wine (n = 10), wine velum (n = 14), and from oak trees (n = 9), as a wild reference.Because SO 2 is an intermediate of the sulfur assimilation pathway, and used in most wine fermentations as an additive for its antimicrobial, antioxidant and anti-oxidizing activities, we compared the H 2 S production of the mentioned ecological groups in a synthetic grape must in the absence or presence of sulfites.The variability in H 2 S production among strains of these three groups is presented in Fig. 1 and Supplementary Fig. 1.A two-way ANOVA revealed that the origin of the strain has a significant effect on H 2 S produced (F 2,128 = 36.31,p_value = 3.24 × 10 -13 ) as well as the addition of SO 2 to the must (F 1,128 = 59.19, p_value = 3.36 × 10 -12 ).A significant interaction between the effects of the two factors (i.e.SO 2 and origin) on H 2 S produced during alcoholic fermentation (F 2,128 = 14.5, p_value = 2.20 × 10 -6 ) was detected.
Tukey multiple comparisons of means at 95% family-wise confidence level showed that H 2 S production between the independent origins was significant when the must contained sulfite (Fig. 1b).Wine strains produced more H 2 S compared to velum and oak (p_value = 1.50 × 10 -6 and 0.014 respectively).Oak isolates also produced more H 2 S than velum strains in the presence of sulfite (p_value = 1.50 × 10 -6 ).This difference was not noticeable when the must did not contain sulfites (Fig. 1a).

Evaluating the relation between H 2 S production and CUP1 copy number
Besides inducing H 2 S production, the copper content in the growth medium controls the expression of CUP1 33 that is involved in its detoxification.In addition, we observed that CUP1 is one of the proteins with the highest sulfur containing aminoacid content (21.31%), just after MNC1 (25.76%), another membrane protein that is upregulated by toxic concentrations of heavy metal ions 34 .
In a first approach aimed at exploring the effect of CUP1 copy number on H 2 S production in strains of the same three niches analysed above, we increased the number of strains to test (+ 18 wine isolates, total n = 51), in order to include strains with 2 to 71 CUP1 copy number.Surprisingly, we observed a non-linear relation between CUP1 copy number and total H 2 S production.
As shown in Fig. 3A, strains with 1 to 10 CUP1 copies exhibit an increasing total H 2 S production, whereas for more copies, it progressively decreases until it reaches almost null values.A 3rd degree polynomial model described well the H 2 S production in relation to the CUP1 copy number of the strains (black line in Fig. 3), displaying a bell shape, that remained even after the removal of the two highest values for H 2 S production (red line in Fig. 3A).A complex polynomial relation between CUP1 copy number and H 2 S production is noticeable when the model was built with wine strains only (Fig. 3C).
This bell shape is conserved and amplified in the presence of sulfite in the media, except for strains with a high copy number of CUP1 (Fig. 3B,D).
Notably, the increase in H 2 S concentration with the copy number of CUP1 within the range 1-10 copies is similar to the response caused by the increase in copper content of the grape must observed for VL1 and LMD17.

Impact of the modulation of CUP1 copy number on H 2 S production
In order to validate the effect of CUP1 copy number on H 2 S production, we tried to manipulate the number of CUP1 copies per cell.With this aim, we built a multicopy yeast episomal plasmid (YEp) expressing CUP1 under the control of the strong promoter from the translational elongation factor EF-1 alpha (TEF1).Three strains with different number of genomic copies of CUP1 were transformed with this plasmid and tested in a media containing a low copper concentration (0.25 mg/L).
First, the overexpression of CUP1 in the oak strain OAK-Rom 3_2, a low H 2 S producer with one copy of CUP1, led to a significant increase of H 2 S production (F 2,6 = 9.61 p_value = 0.013, Fig. 4), without affecting the growth.In contrast to the oak strain, the overexpression of CUP1 in the wine strain LMD17, a highH 2 S producer, with 11 copies of CUP1, decreased H 2 S production by half (F 2,5 = 17.49, p_value = 0.006, Fig. 4), with no impact on fermentation kinetic.Last, the overexpression of CUP1 in L1374, which carries 36 copies of CUP1 and was ranked among the lowest H 2 S producers, did not change its production (F 2,6 = 0.2, p_value = 0.824, Fig. 4).The responses displayed by these three constructions are in agreement to the experimental data presented in Fig. 3, reproducing the "bell-shape" trend of H 2 S production.

Impact of sulfite addition in the culture media on copper resistance
Because sulfite is required for the synthesis of sulfur containing amino acids essential for CUP1 synthesis, it was logical to test how the deregulation of the pathway by exogenous sulfite might affect copper resistance.Three strains were tested: oak strain Oakrom 3.2, and wine strains LMD17 and L1374, that have 1, 10 and 34 copies of CUP1 respectively.The overexpression of CUP1 in Oakrom 3.2, increased the resistance to copper in the control media, which was not the case for wine strains LMD17 and L1374.However, yeast growth was improved for all strains overexpressing CUP1 on copper-supplemented media when SO 2 was present (Fig. 5A,B).

Discussion
Our findings, obtained under conventional winemaking conditions, involving the presence of sulfites, demonstrate for the first time that the production of hydrogen sulfide (H 2 S) by Saccharomyces cerevisiae during alcoholic fermentation varies among natural and two distinct groups of domesticated strains.Surprisingly, wine populations exhibited the highest H 2 S production levels when sulfites are added to the grape must.This observation is unexpected, given the widely acknowledged undesirability of H 2 S in winemaking processes, caused by its unpleasant smell of rotten egg.Indeed, high residual concentrations of H 2 S require specific treatment to eliminate this off-flavour.The increase in H 2 S production induced by sulfite addition is explicable due to its role as an intermediate metabolite in the sulfur assimilation pathway.However, the significant differences we observed between wine and oak strains are intriguing, especially considering that wine strains harbour several  types of translocations leading to a higher expression of the sulfite efflux pump SSU1.This apparently contradicts the antagonistic role of SSU1 in copper resistance 30 , suggesting a limitation in the sulfur assimilation pathway.However, it should be noted that Onetto et al.'s study was conducted in the absence of added sulfites and indeed, we could show that the introduction of sulfites into the grape must, as commonly practiced by winemakers, increases copper resistance, including for strains with a high CUP1 copy number.We therefore propose that exogenous sulfite may exceed the expulsion capacity of the sulfite export transporter, thereby increasing H 2 S, and hence sulfur containing amino acids and CUP1 synthesis in wine strains.
We also demonstrate that the presence of copper in grape must increases H 2 S production.Our results align with previous expression data 35,36 , which revealed that copper exposure triggers a higher expression of genes encoding the two subunits of the sulfite reductase MET5, MET10 (i.e., the main enzyme of the sulfur assimilation pathway), and the protein responsible for copper resistance/detoxification CUP1 following.Furthermore, the metallothionein protein Cup1p, which has one of the highest contents of sulfur-containing amino acids methionine and cysteine in the S. cerevisiae proteome, requires the availability of these amino acids for its synthesis.The differences in H 2 S production among oak, wine, and velum yeast strains may be attributed to variations in the number of CUP1 copies in their genomes.However, we describe a complex relationship between the number of CUP1 copies and H 2 S production.Moderate amplification of CUP1 (up to approximately 10 copies) leads to an increase in H 2 S production, whereas higher copy numbers result in a lower fraction of H 2 S being stripped by the CO 2 generated during fermentation.One possibility of this curve could result from an increasing activation of SAP to support CUP1 production when up to at least 10 gene copies are present, whereas for higher number of copies, the higher requirement of sulfur amino acids could exceed maximum activity of the SAP, leading to an increased use of H 2 S for the synthesis and a lower release.The increased resistance obtained in the presence of SO 2 supports this hypothesis.In this case, we propose that this reflects a higher utilization of H 2 S for amino acid synthesis.
Lastly, our results also shed light on the specific behaviour of flor strains.Unlike wine strains, velum strains exhibit very low H 2 S production, and have a lower number of CUP1 copies.Velum strains grow at the surface of wine, after alcoholic fermentation, which significantly reduces the copper content of wine.It is likely that the selection pressure for copper-resistant strains has been less intense for flor strains compared to wine yeast.Another possible explanation for the lower H 2 S production in velum strains is the reduced activity of the pentose phosphate pathway, which provides NADPH, in comparison to oak, bread and wine strains 37 .This observation elucidates the divergent domestication trajectories of wine and flor strains, reflecting their distinct lifestyles 13 .

Conclusion
The long-term exposure of yeast to copper, used for vine pest management over 150 years, has led to their adaptation by selecting strains with multiple copies of CUP1.Our results suggest that this adaptation involves a significant trade-off: increased resistance to copper, but also high H 2 S production by the yeast, which is detrimental to wine quality.This increased H 2 S production is further exacerbated in the presence of sulfite, another common additive in winemaking.Given the energetic cost of H 2 S production, its impact on the global yeast metabolism should be evaluated.Although many projects and techniques have been dedicated to understanding and limiting H 2 S production 26,27,28 , none have investigated the potential role of copper use in causing this phenotype.Therefore, the yeast CUP1 background should be considered when selecting wine yeast for low H 2 S production.However, diversity data suggests that the amplification of CUP1 likely is not the sole mechanism explaining variations in H 2 S production, which requires further investigations.

Strains
Fifty-one Saccharomyces cerevisiae from different geographical areas were characterized for their H 2 S production during alcoholic fermentation.The genetic group, identified in previous works indicated in the references of Supplementary Table 1, reflected the colonized ecological niche: 28 belong to the "wine" clade, 14 to "velum" group and 9 to the "oak" one.Strains were selected from our laboratory collection and maintained on solid medium (agar YPD: 2% glucose, 1% yeast extract, 2% bactopeptone, 2% agar) at 4 °C.

Fermentation conditions and H 2 S quantification
Fermentation experiments were conducted using synthetic must (SM), designed to mimic the characteristics of a natural grape must 38 .It contained a 200 g/L equimolar glucose and fructose content, and 200 mg/L assimilable nitrogen, 3.8 mg/L phytosterol, and 0.25 mg/L of Cu 2+ .The pH was adjusted to 3.3 with sodium hydroxide solution.
One colony of each strain was grown in 5 ml of liquid YPD at 28 °C for 24 h and then diluted 100 times in SM.After 24 h at 28 °C, cells were counted with an electronic particle counter (Multisizer 3 counter; Beckman Coulter) and 250 mL of SM, supplemented with 60 mg/L of SO 2 when the impact of sulfite was evaluated, were inoculated to 1 × 10 6 cells/mL.Fermentations were carried out at 28 °C, under permanent stirring (280 rpm) and they were followed daily by weight loss, until the theoretical percentage of sugar consumed reached 95% (87.4 g CO 2 /L produced).Total H 2 S produced during alcoholic fermentation was collected with a zinc-based trap system and quantified with sulfide specific fluorescent probe, as described before 32 .
When the impact of the overexpression of CUP1 was in study, SM was supplemented with Geneticin (G418-Sigma A1720-5G) to maintain the plasmid allowing the overexpression itself.Suitable antibiotic concentrations were defined for each strain (100 µg/mL for wine strains, 40 µg/mL for the oak one), to simultaneously allow the maintenance of the plasmid and a good fermentation rate, but prevent the growth of the sensitive strain (i.e. the wild-type strain without the plasmid).www.nature.com/scientificreports/When assessing the impact of copper concentration on H 2 S production, SM was supplemented with copper sulfate to reach 1 or 2 mg/L of copper; control copper concentration was 0.25 mg/L in all the experiments.More details about the experiments are given in the "Experimental design and statistical analyses" section.

Drop test on copper and sulfite supplemented media
Copper resistance in presence or absence of SO 2 , was assessed by a drop-test for three wild type strains with different CUP1 copy number in their genome (Oak-Rom 3-2, LMD17 and L1374), and their counterpart engineered to over-express CUP1 (see below).Triplicates of these strains were grown overnight at 28 °C in 5 mL of YPD.Cells were then counted with an electronic particle counter (Multisizer 3 counter; Beckman Coulter), washed with PBS and resuspended in sterile PBS to obtain 10 7 cells/mL.Three successive 1/10 dilutions were prepared and 1.5 µL of each dilution was spotted on synthetic must having the same composition of the one used for the fermentations, gelled with 20 g/L agar.According to the tested modalities, copper (0, 0.5, 1, 6, 12 mM) and sulfite (0, 40, 60 mg/L SO 2 ) were added to the media to evaluate their effect.Agar plates were incubated at 28 °C for 72 h and growth was assessed by visual examination.

CUP1 copy number evaluation
For most of the strains, CUP1 copy number was estimated from their genome sequence, obtained from previous works or from sequencing performed in this study.To obtain the values, the median sequencing depth measured at SNPs encountered between coordinates 212,500 and 213,000, and between 214,500 and 215,000 on Chromosome VIII was divided by the median sequencing depth over the entire genome (excluding mitochondria and 2 microns).For Italian strains, CUP1 copy number data had been already quantified by Real Time PCR 18 .
DNA purity was checked from the 260 nm/280 nm and 260 nm/230 nm OD ratio measured with NanoDrop 1000 (ThermoScientific).The DNA was quantified by fluorescence using the QuantiFluor kit, dsDNA system (Promega) and then stored at − 20 °C.

Genome sequence and analysis
DNA samples were processed to generate libraries of 500 bp inserts.After passing quality control, the libraries were sequenced with DNBseq technology using BGISEQ-500 platform, generating paired-end reads of 2 × 150 bp.
Reads were then mapped to the S288C reference genome with BWA v0.6.2 with default parameters 41 and genotyping made with samtools v1.11 to obtain a variant file including the sequencing depth of each variant position.Sequence positions were afterwards filtered for quality criteria: sufficient coverage position as well as genotyping and mapping quality (MQ > − 20) were kept.

Plasmid construction and yeast transformation
CUP1 was inserted via Gibson assembly method 42 between TEF promoter and terminator in a high copy Yeast Episomal plasmid (YEp352), modified to confer geneticin resistance (YEp352-G418) to the host cell.In detail, the backbone was amplified with primers P1 and P2, designed to replace the original URA3 copy of YEp with CUP1, since the strains used were not auxotrophic and the selection had been made by antibiotic.Therefore, the backbone contained a 2 um replication origin (multicopy), AmpR, ColE1, pPGK and G418 resistance cassette.CUP1 was amplified from OakGri7_1, a strain previously sequenced by our laboratory 10 , with a single metallothionein copy and the same sequence as laboratory reference strain S288C, used to design primers (P5-P6).TEF promoter and terminator were amplified from pCfB2312 43 with primers P3-P4 and P7-P8, respectively.Primer sequences are listed in Table 1.
Proper fragment insertion was verified by enzymatic digestion (NarI, ClaI, PacI-New England Biolabs).To assure that the phenotype was related to the overexpression of CUP1, a Yep352-G418 plasmid without CUP1 was used as control.PCRs were performed with Phusion™ High-Fidelity DNA Polymerase and validated by gel electrophoresis.Escherichia coli strain DH5α was used to maintain and amplify the plasmid; cells were selected on LB medium with ampicillin (100 µg/mL) and grown at 37 °C.Yeasts (Oak-Rom 3-2, LMD17 and L1374) were transformed with the lithium acetate method 44 and strains containing the recombinant plasmids were selected on YPD agar with 200 μg/mL geneticin (G418-Sigma A1720-5G).
Vol:.( 1234567890 where Yijk is the H 2 S production, µ the overall grand mean, αi is the fixed strain group effect, βj is the fixed SO 2 effect, γij is their interaction effect, and εijk the residual error.The analysis of the residuals showed that three values were distant from the global distribution.Since results of the statistical analysis did not change after removing all the observations of the three outlier strains, the complete dataset was kept as the method is sufficiently robust to mild deviations.

Experiment 2: impact of copper content of the media on H 2 S production
Fermentations were performed without SO 2 in triplicate, for each strain (VL1 and LMD17) and each condition (0.25-1 and 2 mg/L of copper).
To evaluate the effect of copper and strains on H 2 S production, ANOVA was performed, after checking for the equality of variance with a Levene test.The most parsimonious model was kept after checking of the absence of interaction between strain and the copper content: where Yijk is the H 2 S production, µ the overall grand mean, αi is the fixed strain effect, βj is the fixed copper effect and εijk the residual error.

Experiment 3: impact of CUP1 copy number on H 2 S production
To the strains evaluated in experiment 1, we added 18 wine strains (total strains analyzed = 55), some known to harbor a high number of copies of CUP1, and some commercial strains known to be high H 2 S producers (Supplementary Table 1, Dataset 2).Alcoholic fermentations were performed in absence of SO 2 , in duplicate for each strain.
Different polynomial models were used to describe the interaction between H 2 S production and CUP1 copy number (first-, second-and third-degree polynomial models); ANOVA was used to assess the significance of these models.

Experiment 4: impact of the overexpression of CUP1 on H 2 S production
Fermentations were performed without SO 2 and with standard copper content (0.25 mg/L) in triplicate, for each strain (OAK_ROM 1-3, LMD17 and L1374) and each condition (wild-type strain, strain with the empty vector, strain with the CUP1 overexpressing vector).
ANOVA was performed to test the effect of the genetic modification in each strain.The model used was: where Yij is the H 2 S production, µ the overall grand mean, αi is the fixed genetic modification effect, and εij the residual error.Figure 6 summarizes the experimental design.For all the experiments, when the impact of one (or more) factor was significant, differences between modalities were evaluated by post-hoc testing (Tukey's HSD multiple-comparison test, p < 0.05).

Compliance with international and national regulation
Yeast strains were available from culture collection, or gifted by other authors, or provided by the company Lallemand.The yeast collection and use was in accordance with all the relevant guidelines.

Figure 1 .
Figure 1.Difference in the average of cumulate H 2 S production during alcoholic fermentation of velum, oak and wine strains in absence (A) or presence (B) of SO 2 .

Figure 2 .
Figure 2. Effect of copper content in synthetic must without SO 2 on total H 2 S production during alcoholic fermentation by wine strains VL1 and LMD17.p values refer to two-way ANOVA.

Figure 3 .Figure 4 .
Figure 3. H 2 S production (in synthetic must without SO 2 ) distribution as function of CUP1 copy number of each strain.(A) Whole set of studied strains, in synthetic must without SO 2 ; (B) whole set of studied strains, in synthetic must supplemented with SO 2 ; (C) wine strains, in synthetic must without SO 2 ; (D) wine stains in synthetic must supplemented with SO 2 ; Isolation origins are described by colours as in Fig. 1.Black solid line: polynomial model describing the relation between H 2 S production and CUP1 copy number; red line: same model excluding the two highest H 2 S producers.

Table 1 .
Primers used in this work.